Fluidics system for sequential delivery of reagents

ABSTRACT

The invention provides a passive fluidics circuit for directing different fluids to a common volume, such as a reaction chamber or flow cell, without intermixing or cross contamination. The direction and rate of flow through junctions, nodes and passages of the fluidics circuit are controlled by the states of upstream valves (e.g. opened or closed), differential fluid pressures at circuit inlets or upstream reservoirs, flow path resistances, and the like. Free diffusion or leakage of fluids from unselected inlets into the common outlet or other inlets at junctions or nodes is prevented by the flow of the selected inlet fluid, a portion of which sweeps by the inlets of unselected fluids and exits the fluidics circuit by waste ports, thereby creating a barrier against undesired intermixing with the outlet flow through leakage or diffusion. The invention is particularly advantageous in apparatus for performing sensitive multistep reactions, such as pH-based DNA sequencing reactions.

This is a continuation of U.S. patent application Ser. No. 13/245,649,filed 26 Sep. 2011, which is a continuation of U.S. patent applicationSer. No. 12/785,667, filed 24 May 2010, now U.S. Pat. No. 8,546,128,which is a continuation-in-part of U.S. patent applications Ser. Nos.12/474,897 (which claims priority under U.S. provisional applicationSer. Nos.: 61/205,626, filed Jan. 22, 2009; 61/198,222, filed Nov. 4,2008; and, 61/196,953, filed Oct. 22, 2008) and Ser. No. 12/475,311,both filed 29 May 2009, and claims priority under U.S. provisionalapplication Ser. No. 61/291,627 filed 31 Dec. 2009. Each of theforegoing applications is incorporated by reference in their entireties.

BACKGROUND

Many applications require the regulation of multiple fluid flows in amanner that minimizes intermixing or cross-contamination of thedifferent fluids. Such applications include multi-step synthetic oranalytical processes that are carried out in a common volume and thatcomprise successive cycles of reagent delivery using fluids fromseparate reservoirs. e.g. Margulies et al. Nature, 437: 376-380 (2005);Merrifield et al, U.S. Pat. No. 3,531,258; Caruthers et al, U.S. Pat.No. 5,132,418; Rothberg et al, U.S. patent publication 2009/0127589, andthe like. Although fluidics systems are available for selectivelyswitching multiple reagent solutions to a common chamber for processing,they suffer from several deficiencies, including but not limited to, thepresence of large surface areas that can adsorb or retain reagents,large physical size which makes it difficult to use with miniaturizedfluidics components, e.g. see Rothberg et al (cited above), lessaccessible surfaces including edges and/or corners which make completepurging and removal of successive reagents difficult or inefficient, andthe use of moving parts which can wear out and lead to highermanufacturing and assembly costs, e.g. Hunkapiller, U.S. Pat. No.4,558,845; Wittmann-Liebold et al, U.S. Pat. No. 4,008;736; Farnsworthet al, U.S. Pat. No. 5,082,788; Garwood et al, U.S. Pat. No. 5,313,984;or the like.

In view of the above, it would be advantageous to have available adevice for regulating multiple fluid flows to a common volume forcomplex synthetic or analytical processes which overcame thedeficiencies of current approaches.

SUMMARY OF THE INVENTION

The present invention is directed to apparatus and methods fordelivering multiple fluids to a common volume, such as for example, apassage or conduit to a reaction chamber or flow cell. The inventionalso includes applications of such apparatus and methods in multistepanalytical or synthetic processes. The present invention is exemplifiedin a number of implementations and applications, some of which aresummarized below and throughout the specification.

In one aspect, the invention provides a passive fluidics circuit forsequentially directing different fluids to a common volume, such as areaction chamber or flow cell, without intermixing or crosscontamination. As used herein, such sequential directing is sometimesreferred to as “multiplexing” a plurality of fluid flows. The directionand rate of flow through junctions, nodes and passages of the fluidicscircuit are controlled by the states of upstream valves (e.g. opened orclosed), differential fluid pressures at circuit inlets or upstreamreservoirs, flow path resistances, and the like. Free diffusion orleakage of fluids from unselected inlets into the common outlet or otherinlets at junctions or nodes is prevented by the flow of the selectedinlet fluid, a portion of which sweeps by the inlets of unselectedfluids and exits the fluidics circuit by way of waste ports, therebycreating a barrier against undesired intermixing with the outlet flowthrough leakage or diffusion. In one aspect, the selected fluidic inletprovides a laminar flow of fluid through the fluidics node.

In another aspect, the invention provides a fluidics circuit forcontrolling a plurality of fluid flows, the fluidics circuit comprising:(a) a fluidics node having an outlet and a plurality of fluid inlets;and (b) at least one waste port in fluid communication with the fluidicsnode by one or more passages each having a fluid resistance, the fluidresistances of the passages being selected so that whenever a fluidflows solely through a single fluid inlet to form a flow in the fluidicsnode a portion of such fluid exits the fluidics node through the outletand the remainder of such fluid exits the fluidics node through the oneor more passages, such that any fluid entering the fluidics node frominlets without fluid flows (i.e. “unselected inlets”) is directedthrough the one or more passages to the one or more waste ports. In oneembodiment, the plurality of fluid flows is controlled to provide apredetermined sequence of fluid flows through the outlet of the fluidicsnode. In another embodiment, such control is implemented by valves anddifferential pressures applied to the fluids of the flows upstream ofthe fluidics circuit.

In another aspect, the invention provides a fluidics circuit with nomoving parts that sequentially directs multiple fluids to a commonvolume with no intermixing. Since the fluidics circuit comprises only anode and a plurality interconnected passages where fluid movement iscontrolled by remotely positioned valves, pumps, it can be readilyminiaturized by conventional microfluidics techniques for applicationswhere size and mass are critical factors. Furthermore, the use of thefluidics circuit for fluid switching without the use of impermeablebarriers makes the circuit ideal for use in processes where a stablereference potential is required, such as in electrochemical processes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration of one embodiment of the invention which hasinlets and an outlet on opposing surfaces of a fluidics node.

FIGS. 1B-1D illustrate priming, reagent flow, and wash steps in anembodiment of the invention.

FIG. 2 illustrates another embodiment of the invention where a singleresistive passage connects a waste port with a plurality of inlets.

FIGS. 3A-3C illustrate another embodiment of the invention where each ofa plurality of inlets is connected to a central fluidics node and awaste port through a planar network of passages.

FIGS. 4A-4B illustrate another embodiment having a planar structure thatmay be duplicated by stacking similar units that are connected throughtheir fluidics nodes and waste passages. thereby enabling theaccommodation of more input fluids.

FIG. 5 illustrates how the fluidics circuit of the invention may providea stable reference electrode for a multi-step electrochemical process.

FIG. 6 is a diagrammatic illustration of an exemplary apparatus using afluidics system of the invention.

FIG. 7 illustrates an embodiment providing separate wash control in adual-flow chamber flow cell that provides an uninterrupted fluid pathbetween a reference electrode and both chambers of the flow cell.

DETAILED DESCRIPTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of mechanicalengineering, electronics, fluid mechanics, and materials science, whichare within the skill of the art. Such conventional techniques include,but are not limited to, design and fabrication of fluidics andmicrofluidics devices, and the like. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.

The invention provides methods and apparatus for rapidly and cleanlyswitching flows of different fluids to a common outlet using a fluidicscircuit. In one aspect, the fluidics circuit of the invention iscombined with fluidic reservoirs, valves, pressure sources, pumps,control systems, and/or like components, to form a fluidics system fordelivering separate fluid flows having predetermined rates and durationsto a common volume, such as an outlet, chamber, flow cell, or the like.Such fluidics circuits are particularly useful in fluidics systems inapparatus for carrying out multi-step chemical, enzymatic, orelectrochemical processes, such as described Margulies et al, Nature,437: 376-380 (2005); Merrifield et al. U.S. Pat. No. 3,531,258; Brenneret al, Nature Biotechnology, 18: 630-634 (2000); Ronaghi et al, Science,281: 363-365 (1998); Caruthers et al, U.S. Pat. No. 5,132,418; Namsaraevet al, U.S. patent publication 2005/0100939; Rothberg et al, U.S. patentpublication 2009/0127589; and the like.

In one aspect, the fluidics circuit of the invention provides a junctionwhere a flow of a selected fluid is split into at least two branches:one branch is directed to an outlet and from there to a flow cell orreaction chamber for use and the other branch is directed past theunselected fluid inlets and from there away from the outlet and to awaste port. In one embodiment, such flows are created by balancing thefluid resistance of the fluid outlet and that of the one or morepassages between the fluid inlets and the waste port. Preferably, theflow rates, fluid viscosities, compositions, and geometries and sizes ofthe passages, chambers and nodes are selected so that fluid flow islaminar within the fluidics circuit. Guidance for making such designchoices is readily available from conventional treatises on fluiddynamics, e.g. Acheson, Elementary Fluid Dynamics (Clarendon Press,1990), and from free or commercially available software for modelingfluidics systems, e.g. SolidWorks from Dassault Systems (Concord.Mass.); Flowmaster from Flow Master USA, Inc. (Glenview, Ill.); and OpenFOAM (open source code for computational fluid dynamics available on theworld wide web, www.openefd.co.uk). Fluidic circuits and apparatus ofthe invention are particularly well suited for meso-scale andmicro-scale fluidics systems, for example, fluidics systems havingpassage cross-sections in the range of tens of square microns to a fewsquare millimeters, or having flow rates in the range of from a fewnL/sec to a hundreds of μL/sec. The number of fluid flows controlled byfluidics circuits of the invention can vary widely. In one aspect,fluidics circuits of the invention control a plurality of flows in therange of from 2 to 12 different fluids, or in another aspect in therange of from 2 to 6 different fluids.

Fluidics Circuits

The design and operation of one embodiment of the invention is partiallyillustrated in FIG. 11A. Four fluid inlets, or reagent inputs, (100,102, 104, 106) are connected to fluidics node (108) and are in fluidcommunication with, and on an opposing surface to outlet (110). Valve(111) is shown open so that fluid passes through inlet (100) intofluidics node (108). A portion (124) of the fluid travels through apassage shown on the left, a portion (126) travels through a passageshown on the right, and a portion exits the fluidics node through outlet(110). Preferably the three fluid flows are laminar and the flow alongthe surface containing the fluid inlets exits the fluidic node in aperiod of time that is much less than the time it would take materialfrom the unselected inlets (diffuse effluent (128)) to diffuse to theopposing surface of the fluidics node. In this way, intermixing of thedifferent input reagents that exit through outlet (110) is avoided. Inone mode of operation, reagent inputs are selected by opening the valvecorresponding to such reagent and closing all the other valves. Asillustrated in this embodiment, valve (111) is open and valves (113,115, and 117) are closed. In the closed state, even though there is noflow in the unselected inlets, a volume (for example, 120) of theunselected fluid is in free diffusive contact with the selected fluid.The split laminar flow of the selected fluid to both outlet (110) andpast the unselected inlets and to the waste ports prevents undesiredmixing. FIGS. 1B-1D further illustrate the operation of the aboveembodiment. As in FIG. 1A, inlets (100, 102, 104, and 106) connect tofluidics node (108) on a surface opposite of outlet (110) and passages(130 and 132) connect fluidics node (108) to waste port (134). Thelength (136) and width (138) of passages (130 and 132) are selected toprovide fluid resistance so that the flow of fluid from an inlet isbalanced between fluid exiting the node through outlet (110) and fluidexiting the node through passages (130 and 132). Also illustrated iswash fluid inlet (140) which is connected to outlet (110) and in fluidcommunication with fluidics node (110). In one mode of operation,referred to as “prime reagent” mode, wash inlet valve (not shown) isopened and valve (not shown) of reagent inlet (104) is opened. A washsolution flows into outlet (110) and towards an application, e.g. a flowcell containing a chip, as described in Rothberg et al (cited above),and towards fluidics node (108) where it combines with and constrainsfluid from inlet (104) to flow into waste port (134). Exemplary flowrates and times are listed in the figure for a particular applicationdescribed more fully below, but generally such rates and time are designchoices that depend on a particular application. In another mode ofoperation, referred to in FIG. 1C as “flow reagent” mode, the flow ofwash solution is shut off and the sole flow emanates from inlet (104).The flow is split into three branches two traveling through passages(130) and (132) and one traveling through outlet (110). In another modeof operation, referred to in FIG. 1D as “wash” mode valves of all fluidinlets (100, 102, 104 106) are close and the valve of wash inlet (140)is opened so that only wash solution enters the fluidics node (108),passes over inlets (100, 102, 104, 106), and exits through passages(130) and (132).

FIG. 2 illustrates diagrammatically in top and side views anotherembodiment of a fluidic circuit, which uses ring-shaped waste andresistive passages (206 and 208, respectively) to accommodate a greaternumber of inlets (200) than the embodiment of FIGS. 1A-1D. As above,multiple inlets (200) connect to fluidics node (202) in a surfaceopposite to that connecting to outlet (204). Fluid flow from an inlet issplit in fluidics node (202) so that a portion exits outlet (204) andthe remainder exits ring-shaped passage (208), whose width (210) andheight (212) are selected to provide fluidic resistance forappropriately splitting the input reagent flow. After sweeping pastunselected inlets and passing through resistive passage (208), flow froma selected inlet enters waste ring passage (206) and is directed towaste port (214).

FIGS. 3A-3C diagrammatically illustrate another embodiment of thefluidics circuit of the invention which accommodates five input reagentsin a planar circuit structure. FIG. 3A is a top view of a transparentbody or housing (300) containing fluidic circuit (302). Housing (300)may be constructed from a variety of materials, including metals, glass,ceramics, plastics, or the like. Transparent materials includepolycarbonate, polymethyl methacrylate, and the like. Inlets (or inputports) (304, 306, 308, 310, and 312) are connected by a passage to theirrespective connector slots (314) located on the bottom side of housing(300) (shown as double circles concentric with the inlets) from whichreagents enter fluidic circuit (302). Inlets (304, 306, 308,310, and312) are in fluid communication with passages (305, 307, 309, 311, and313, respectively) which, in turn, are connected to curvilinear passages(324, 326, 328, 330, and 332, respectively). Each curvilinear passageconsists of two legs, such as (336) and (338), identified forcurvilinear passage (324) at a “T” junction (335), also identified foronly curvilinear passage (324). One leg is an inner leg (for example(338)) which connects its respective inlet to node (or multi-use centralport) (301) and the other leg is an outer leg (for example (336)) whichconnects its respective inlet to waste passage (or ring) (340). Asmentioned above, the cross-sectional areas and lengths of the inner andouter legs of the curvilinear passages may be selected to achieve thedesired balance of flows at the “T” junctions and at node (301). Throughpassage (344), waste passage (or channel) (340) is in fluidcommunication with waste port (345) which connects to a waste reservoir(not shown) by connector slot (346) on the bottom side of body (300).Node (301) is in fluid communication with port (360) by passage (361)which in this embodiment is external to body (300) and is illustrated bya dashed line. In other embodiments, passage (361) may be formed in body(300) so that connector slots for node (301) and port (360) are notrequired. Port (360) is connected by passage (363) to wash solutioninlet (362), where a “T” junction is formed, and to connector slot (364)which, in turn, provides a conduit to a flow cell, reaction chamber, orthe like. FIGS. 3B and 3C illustrate two of three modes of using thefluidics circuit to distribute fluids to a flow cell. The modes ofoperation are implemented by valves (350) associated with each of theinput reagents and with the wash solution. In a first mode of operation(selected reagent valve open, all other reagent valves closed, washsolution valve closed) (FIG. 3B) a selected reagent is delivered to aflow cell; in a second mode of operation (selected reagent valve open,all other reagent valves closed, wash solution valve open) (FIG. 3C) thefluidic circuit is primed to deliver a selected reagent; and in a thirdmode of operation (all reagent valves closed wash solution valve open)(not shown), all passages in the fluidics circuit are washed. Asmentioned above, associated with each inlet is a valve (350) which canbe opened to allow fluid to enter fluidic circuit (302) through itsrespective inlet (as shown for valve (352)), or closed to prevent fluidfrom entering circuit (302) (as shown with all valves, except for(352)). In each case, when an inlet's valve is open and the others areclosed (including the wash solution valve) as shown for inlet (370) inthe FIG. 3B, fluid flows through passage (354) to “T” junction (356)where it is split into two flows, one of which is directed to wastepassage (340) and then the waste port (345), and another of which isdirected to node (301). From node (301) this second flow again splitsinto multiple flows, one of which exits node (301) through passage (361)and then to passage (363) and to a flow cell and the other flows to eachof the passages connecting node (301) to the other inlets, and then towaste passage (340) and waste port (345). The latter flows pass theother inlets carrying any material diffusing or leaking therefrom anddirecting it to waste port (345). A sequence of different reagents maybe directed to a flow cell by opening the valve of a selected reagentand simultaneously closing the valves of all of the non-selectedreagents and the wash solution. In one embodiment, such sequence may beimplemented by a sequence of operating modes of the fluidics circuitsuch as: wash, prime reagent x₁, deliver reagent x₁, wash, prime reagentx₂, deliver reagent x₂, wash, and so on. The reagent priming mode ofoperation is illustrated in FIG. 3C. As in the reagent delivery mode,all reagent inlet valves are closed, except for the valve correspondingto the selected reagent. Unlike the reagent delivery mode, however, thewash solution valve is open and the relative pressure of the selectedreagent flow and the wash solution flow is selected so that washsolution flows through passage (361) and into node (301) where it thenexits through all the passages leading to waste passage (340), exceptfor the passage leading to the selected reagent inlet.

FIGS. 4A-4B diagrammatically illustrates another embodiment of a planarfluidics circuit which accommodates four input reagents and whose designcan accommodate further input reagents by stacking of the planarfluidics circuit and connecting their fluidics nodes. The topology andoperation of the planar fluidics circuit of FIG. 4A is equivalent tothat of FIG. 3A. except that the latter includes an additional inlet andin the former, flows through “T” junctions (as exemplified by (421)) arebalanced by selecting different cross-sectional areas of the differentlegs (one connecting to node (400) and one connecting to waste channel(415)) of each passage (404, 406, 408, and 410), rather than byselecting legs of different length and/or curvature. Inlets (412, 414,416, and 418) connect to passages (404, 406, 408, and 410, respectively)through “T” junctions, e.g. (421), which, in turn, connect to wastepassage or channel (415) and fluidics node (400). Outlet (402) and wastepassages (424, 426, 428, and 430) connect a stack of planar fluidiccircuits as illustrated in FIG. 4B.

FIG. 5 diagrammatically illustrates how fluidics circuits of theinvention may be used in an electrochemical process requiring multiplereactants, including electrolytes used in such processes, and employinga reference electrode upstream of a reaction chamber. For stablereference voltages, it is desirable that the reference electrode contactno more than a single process reagent. Fluidics circuits of theinvention provide a means of delivering a predetermined sequence ofelectrolytes through a common inlet of a reaction chamber whilemaintaining (i) uninterrupted fluid communication between the reactionchamber and a reference electrode, and (ii) contact of only a singleelectrolyte (i.e. a selected electrolyte) with the reference electrode.All of the other reagent or electrolytes (i.e. the unselectedelectrolytes) never contact the reference electrode. Planar fluidicscircuit (500) as described in FIGS. 4A-4B delivers a sequence ofdifferent reagents to reaction chamber (510) by passage (502). Flows ofwash solution may be directed through passage (504) to “T” junction(512) and back to fluidics circuit (500) and to reaction chamber (510),as described above. A stable reference voltage may be provided toreaction chamber (510) by positioning reference electrode (506) in oradjacent to passage (504). In one embodiment, such reference electrodemay be a metal tube forming a section of passage (504), as isillustrated in FIG. 5. Reference electrode (506) is electricallyconnected to a reference voltage source (508).

In one aspect of the invention, such an apparatus comprises a reactionvessel coupled to an electronic sensor for monitoring products in thereaction vessel: a fluidics system including a fluidics circuit of theinvention for sequentially delivering a plurality of differentelectrolytes including a selected electrolyte to the reaction vessel:and a reference electrode in contact with the selected electrolyte forproviding a reference voltage to the electronic sensor, the referencevoltage being provided without the reference electrode contacting anyunselected electrolytes.

Materials and Methods of Fabrication

As mentioned above, fluidic circuits of the invention may be fabricationby a variety of methods and materials. Factors to be considered inselecting materials include degree of chemical inertness required,operating conditions, e.g. temperature, and the like, volume of reagentsto be delivered, whether or not a reference voltage is required,manufacturability, and the like. For small scale fluid deliveries,microfluidic fabrication techniques are well-suited for making fluidicscircuits of the invention, and guidance for such techniques is readilyavailable to one of ordinary skill in the art, e.g. Malloy, Plastic PartDesign for Injection Molding: An Introduction (Hanser GardnerPublications, 1994); Herold et at, Editors, Lab-on-a-Chip Technology(Vol. 1): Fabrication and Microfluidics (Caister Academic Press, 2009):and the like. For meso-scale and larger scale fluid deliveriesconventional milling techniques may be used to fabricate parts that maybe assembled into fluidic circuits of the invention. In one aspect,plastics such as polycarbonate, polymethyl methacrylate, and the like,may be used to fabricate fluidics circuits of the invention.

Applications in Electrochemical Processes

Fluidics circuits of the invention are useful in electrochemicalprocesses where multiple reagents are delivered to one or more reactorsthat are monitored with electronic sensors requiring a referenceelectrode. Exposure of a reference electrode to multiple reagents canintroduce undesirable noise into the signals detected by the electronicsensors. Circumstances where this occurs are in methods and apparatusfor carrying out label-free DNA sequencing, and in particular, pH-basedDNA sequencing. The concept of label-free DNA sequencing, includingpH-based DNA sequencing, has been described in the literature, includingthe following references that are incorporated by reference: Rothberg etal, U.S. patent publication 2009/0026082; Anderson et al, Sensors andActuators B Chem., 129: 79-86 (2008); Pourmand et al, Proc. Natl. Acad.Sci., 103: 6466-6470 (2006); and the like. Briefly, in pH-based DNAsequencing, base incorporations are determined by measuring hydrogenions that are generated as natural byproducts of polymerase catalyzedextension reactions. DNA templates each having a primer and polymeraseoperably bound are loaded into reaction chambers (such as the microwellsdisclosed in Rothberg et al, cited above), after which repeated cyclesof deoxynucleoside triphosphate (dNTP) addition and washing are carriedout. Such templates are typically attached as clonal populations to asolid support, such as a microparticle, bead, or the like, and suchclonal populations are loaded into reaction chambers. In each additionstep of the cycle, the polymerase extends the primer by incorporatingadded dNTP only if the next base in the template is the complement ofthe added dNTP. If there is one complementary base, there is oneincorporation, if two, there are two incorporations, if three, there arethree incorporations, and so on. With each such incorporation there is ahydrogen ion released, and collectively a population of templatesreleasing hydrogen ions causing very slight changes the local pH of thereaction chamber which is detected by an electronic sensor. FIG. 6diagrammatically illustrates an apparatus employing a fluidics circuitof the invention for carrying out pH-based nucleic acid sequencing inaccordance with Rothberg et al (cited above). Each electronic sensor ofthe apparatus generates an output signal that depends on the value of areference voltage. The fluid circuit of the apparatus permit multiplereagents to be delivered to the reaction chambers with no more than oneof them contacting the reference electrode, thereby removing a source ofnoise from the output signals generated by the sensors. In FIG. 6,housing (600) containing fluidics circuit (602) is connected by inletsto reagent reservoirs (604, 606, 608, 610, and 612), to waste reservoir(620) and to flow cell (634) by passage (632) that connects fluidicsnode (630) to inlet (638) of flow cell (634). Reagents from reservoirs(604, 606, 608, 610, and 612) may be driven to fluidic circuit (602) bya variety of methods including pressure, pumps, such as syringe pumps,gravity feed, and the like, and are selected by control of valves (614),as described above. The foregoing comprises a fluidics system of theinstrument of FIG. 6. Control system (618) includes controllers forvalves (614) that generate signals for opening and closing viaelectrical connection (616). Control system (618) also includescontrollers for other components of the system, such as wash solutionvalve (624) connected thereto by (622), and reference electrode (628).Control system (618) may also include control and data acquisitionfunctions for flow cell (634). In one mode of operation, fluidic circuit(602) delivers a sequence of selected reagents (1, 2, 3, 4, or 5) toflow cell (634) under programmed control of control system (618), suchthat in between selected reagent flows fluidics circuit (602) is primedand washed, and flow cell (634) is washed. Fluids entering flow cell(634) exit through outlet (640) and are deposited in waste container(636). Throughout such an operation, the reactions and/or measurementstaking place in flow cell (634) have a stable reference voltage becausereference electrode (628) has a continuous, i.e. uninterrupted,electrolyte pathway with flow cell (634), but is in physical contactwith only the wash solution.

FIG. 7 illustrates how the fluidics circuit design concepts may be usedto make a plurality of separate flow chambers using a single large flowcell and sensor array, wherein reagent access to each flow chamber isseparately controlled while still maintaining uninterrupted fluidpathways to the reference electrode for all sensors in all the flowchambers. FIG. 7 is a top view of flow cell (700) that has fluidicsinterface member (702) mounted on and is sealingly attached to a housing(not shown) that holds sensor array (704) and defines two flow chambers(703) and (705), each having separate inlets (706 and 708, respectively)and separate diagonally opposed outlets (710 and 712, respectively) thatare each connected to a common source of reagents from a fluidicscircuit via passages 730 and 735 for flow chamber 1 and 732 and 737 forflow chamber 2, and to separate auxiliary wash reservoirs: 722 for flowchamber 1 and 724 for flow chamber 2. Interior walls (714, 716,718 and720) formed by attachment of fluidics interface member (702) to the chiphousing defines the flow paths through flow chambers (703) and (705) andexclude opposing corner regions (750, 751, 752, and 753) from havingcontact with reagents passing through the flow chambers.

When valve (723) is open, wash solution from the auxiliary washreservoir 1 (722) passes through passage (729), through valve (723), topassage (734), and to junction (731), where the flow splits betweenpassage (735) and passage (741). As with the design of the fluidicscircuits described above, the lengths and cross-sections of passages(735) and (734), and the driving forces of the wash solution and reagentare selected so that when valve (723) is open (as shown) solely washsolution enters flow chamber 1 and reagent from the fluidics circuit isdirected solely to waste reservoir (744). When valve (723) is closed,then no wash solution moves in passage (729) and there is no barrier tothe flow of reagent from passage (730), to passage (735), to passage(741), and to flow chamber 1. Likewise, when valve (725) is open, washsolution from the auxiliary wash reservoir 2 (724) passes throughpassage (743), through valve (725), to passage (736), and to junction(745), where the flow splits between passage (737) and passage (747). Asabove, the lengths and cross-sections of passages (736) and (737), andthe driving forces of the wash solution and reagent are selected so thatwhen valve (725) is open solely wash solution enters flow chamber 2 andreagent from the fluidics circuit is directed solely to waste reservoir(744). When valve (725) is closed (as shown), then no wash solutionmoves in passage (743) and there is no barrier to the flow of reagentfrom passage (732), to passage (737), to passage (747), and to flowchamber 2.

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. The present invention is applicableto a variety of sensor implementations and other subject matter, inaddition to those discussed above.

DEFINITIONS

“Microfluidics device” means an integrated system of one or morechambers, ports, and channels that are interconnected and in fluidcommunication and designed for carrying out an analytical reaction orprocess, either alone or in cooperation with an appliance or instrumentthat provides support functions, such as sample introduction, fluidand/or reagent driving means, temperature control, detection systems,data collection and/or integration systems, and the like. Microfluidicsdevices may further include valves, pumps, and specialized functionalcoatings on interior walls, e.g. to prevent adsorption of samplecomponents or reactants, facilitate reagent movement by electroosmosis,or the like. Such devices are usually fabricated in or as a solidsubstrate, which may be glass, plastic, or other solid polymericmaterials, and typically have a planar format for ease of detecting andmonitoring sample and reagent movement, especially via optical orelectrochemical methods. Features of a microfluidic device usually havecross-sectional dimensions of less than a few hundred square micrometersand passages typically have capillary dimensions, e.g. having maximalcross-sectional dimensions of from about 500 μm to about 0.1 μm.Microfluidics devices typically have volume capacities in the range offrom 1 μm to a few nL, e.g. 10-100 nL. The fabrication and operation ofmicrofluidics devices are well-known in the art as exemplified by thefollowing references that are incorporated by reference: Ramsey, U.S.Pat. Nos. 6,001,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al.U.S. Pat. Nos. 5,126,022 and 6,054,034; Nelson et al, U.S. Pat. No.6,613,525; Maher et al, U.S. Pat. No. 6,399,952: Ricco et al,International patent publication WO 02/24322: Bjornson et al,International patent publication WO 99/19717; Wilding et al, U.S. Pat.Nos. 5,587,128; 5,498,392; Sia et at, Electrophoresis, 24: 3563-3576(2003); Unger et al. Science, 288: 113-116 (2000); Enzelberger et al,U.S. Pat. No. 6,960,437.

What is claimed is:
 1. A fluidic device comprising: a node having afluid outlet; a plurality of fluid inlets, each fluid inlet of theplurality of fluid inlets uniquely associated with a respective reagentof a plurality of reagents; at least one waste port; a plurality ofpassages, each passage of the plurality of passages providing a distinctfluid pathway between the node and the at least one waste port, eachpassage of the plurality of passages including a junction to arespective inlet passage providing fluid communication between the eachpassage and a respective fluid inlet of the plurality of fluid inlets;an outlet passage in fluid communication with the node through the fluidoutlet; and a wash fluid inlet in fluid communication with the outletpassage.
 2. The fluid device of claim 1, wherein each reagent of theplurality of reagents is stored in a respective reservoir.
 3. The fluiddevice of claim 1, further comprising a reference electrode inelectrical communication with the wash fluid inlet.
 4. The fluid deviceof claim 1, wherein the wash fluid inlet includes a branch channelconnected to the outlet passage, the reference electrode disposed in thebranch channel.
 5. The fluid device of claim 1, wherein the outletpassage is in fluid communication with a flow cell.
 6. The fluid deviceof claim 5, further comprising a reference electrode having a continuouselectrolyte pathway through the outlet passage to the flow cell.
 7. Thefluid device of claim 1, wherein each distinct fluid pathway is free ofimpermeable barriers.
 8. The fluid device of claim 1, wherein eachpassage includes a first segment extending from the junction to the nodeand a second segment extending from the junction away from the node. 9.The fluid device of claim 1, wherein the second segment is connected toa waste passage in fluid communication with the waste port.
 10. Thefluid device of claim 1, wherein the second segment has greater flowresistance than the first segment.
 11. A fluidics circuit comprising: atleast three fluid inlets; at least one waste port; a fluid outlet; anode in fluid communication with the fluid outlet; and at least threefluid passages, each fluid passage of the at least three fluid passagesincluding a junction in unique fluid communication with a fluid inlet ofthe at least three fluid inlets, each fluid passage of the at leastthree fluid passages providing a distinct fluid pathway between the nodeand the at least one waste port;
 12. The fluidics circuit of claim 11,wherein the at least three fluid passages are free of valves.
 13. Thefluidics circuit of claim 11, wherein each distinct fluid pathway isfree of impermeable barriers.
 14. The fluidics circuit of claim 11,further comprising a wash fluid inlet in fluid communication with thefluid outlet opposite the node.
 15. The fluidics circuit of claim 14,further comprising a reference electrode in electrical communicationwith the wash fluid inlet.
 16. The fluidics circuit of claim 14, whereinthe wash fluid inlet includes a branch passage connected to the fluidoutlet, the reference electrode disposed in the branch passage.
 17. Thefluidics circuit of claim 14, further comprising a reference electrodehaving a continuous electrolyte pathway through the outlet passage to aflow cell.
 18. The fluidics circuit of claim 11, wherein each reagent ofthe plurality of reagents is stored in a respective reservoir.
 19. Thefluidics circuit of claim 11, wherein the outlet passage is in fluidcommunication with a flow cell.
 20. The fluidics circuit of claim 11,wherein each fluid passage of the at least three fluid passages includesa first segment extending from the junction to the node and a secondsegment extending from the junction away from the node, wherein thesecond segment has greater flow resistance than the first segment.